This renewal application builds upon significant advances in identifying both the genetic basis for autoimmune diseases and discoveries related to the function of costimulatory pathways. As part of this PPG we have defined the immunologic roles of critical costimulatory pathways implicated in the etiology of human autoimmune diseases, focusing on MS and T1D, allowing the identification and molecular characterization of a number of critical costimulatory pathways including B7/CD28, CTLA-4, CD226, ICOS, PD-L1/2 and CD2/CD58 family molecules. The application of genome wide scans to the investigation of these diseases has finally allowed a mechanistic assessment of critical costimulatory pathways based on the unbiased identification of gene variants associated with disease risk. Considering these findings, we will continue to investigate allelic variants and their pathways that are both common (CD226/TIGIT) and unique (CTI_A4-T1D and CDS-MS) to these diseases exploring the underlying hypothesis of this grant that allelic variants in multiple costimulatory pathways lead to a shift from regulatory to inflammatory T cell function. We will explore the costimulatory and signaling pathways identified from genome wide association scans in relationship to regulatory T cell and effector function, investigating the PTPN22, CTLA-4/CD28, CD226/TIGIT, and CDS pathways. This will be accomplished by three approaches: first, we will investigate the immunologic function of these allelic variants in healthy subjects who are not burdened with the inflammatory consequences of autoimmune disease; second, we will directly study patients with MS and T1D, applying our new understanding of disease pathways leading to disease risk in attempts to develop new therapeutic approaches; third; we will study the functional impact of these pathways in mouse models, focusing on how these pathways regulate the functions of effector and regulatory T cells. These goals will be accomplished with four Projects from Harvard and Cambridge Universities with an Administrative Core. RELEVANCE (See instructions): Autoimmune diseases are complex genetic disorders where many common allelic variations lead to disease risk. This understanding is key to developing new therapies. We have elucidated key pathways by performing association scans in patients with MS and Tl D. This grant will translate those genotypic findings to a fundamental understanding of immune phenotypes to identify pathways for future drug targeting. PROJECT 1: Title: - Fuctional Analyses Of Autoimmune Disease Variants Project Leader: Wicker, L PROJECT 1 DESCRIPTION (provided by applicant): PROJECT SUMMARY (See instructions): The project, Functional Analyses of Autoimmune Disease Variants, is focused on three type 1 diabetes (T1D) susceptibility loci, CTLA4, PTPN22 and CD226 in humans and in mice. T1D is a major disease of children with an unexplained steady rise in incidence and increasing numbers of children diagnosed under age 5 years. The research proposed is fully integrated into the PPG activities and goals, and provides a platform to continue the highly productive, interdependent and synergistic collaboration amongst the Pis in Cambridge and Boston aimed at understanding the biological effects of T1D and autoimmune disease gene variants (that we have identified using genetic mapping). T l D gene variants will be studied ex vivo using fresh blood samples from a major resource of genetically-selectable, local healthy volunteers (the Cambridge BioResource) and in vivo, in precisely engineered NOD mouse models of T1D. The Ctla4, Ptpn22 and Cd226 KO alleles will be used to develop NOD strains to model human T1D. A mouse Ptpn22 variant that increases T1D will be a focus of mechanistic studies on the PTPN22 gene, which is part of a molecular pathway that affects multiple human autoimmune diseases. T cells having the susceptibility allele at Ptpn22 have a higher threshold of activation and at a population level fewer of the cells produce IL-2 when stimulated ex vivo. Since both human and mouse gene variants in the IL-2 pathway affect T1D susceptibility, experiments to study gene-gene interactions between the PTPN22 and IL-2 pathways in both species are proposed. Preliminary data indicate that the susceptibility allele at CTLA4, which decreases the expression of soluble CTLA-4, reduces the probability that Tregs will be activated. Overexpression of soluble CTLA-4 in primary T cells and cell lines will be one approach used to study the mechanism by which soluble CTLA-4 affects early events in T cell activation. Since variants at both the PTPN22 and CTLA-4 genes are proposed to alter T cell activation, gene-gene interactions between the PTPN22 and CTLA-4 pathways in both humans and mice will be investigated. RELEVANCE (See instructions): The mortality, morbidity and healthcare costs of type 1 diabetes are enormous. The identification of the genes and pathways that cause type 1 diabetes will allow the identification of early, inherited immunophenotypes or disease precursors that precede autoimmunity and that may be suitable targets or read-outs in ongoing or future clinical trials.